Engineered <i>Bacillus subtilis</i> regulates intestinal barrier in ETEC K88‐infected piglets and modulates miR‐1250‐5p/NFκB pathway in IPEC‐J2

Abstract The substantially increased prevalence of diarrhea in infants and livestock induced by enterotoxigenic Escherichia coli (ETEC) infection threatens the intestinal health of humans and livestock. This study investigated an engineered Bacillus subtilis WB800_KR32 strain expressing antimicrobial peptide KR32 (yielding 2.24 μg/mL with peak activity at 33 h fermentation) for its preventive efficacy against ETEC K88‐induced diarrhea in a piglet model. Oral administration of WB800_KR32 could significantly improve intestinal morphology and barrier function, particularly in the ileum, reduce Escherichia relative abundance, and enhance repair and metabolism‐related pathways while increasing serum antioxidants such as glutathione. Mechanistically, WB800_KR32 significantly decreased novel‐ssc‐miR‐1250‐5p expression and its predicted target, nuclear factor kappa‐B (NFκB) p65 ( p &lt; 0.05). In vitro, 40% fermented WB800_KR32 supernatant reduced novel‐ssc‐miR‐1250‐5p expression, improved tight junction function, decreased inflammatory response, and reduced NFκB p65 phosphorylation in ETEC K88‐infected IPEC‐J2 cells ( p &lt; 0.05). These findings suggested that engineered Bacillus subtilis WB800_KR32 provides a dual‐action therapeutic platform combining targeted antimicrobial delivery with host immunomodulation, offering a promising translational strategy for preventing ETEC K88‐induced diarrhea in humans and livestock.